Plant producing human enterokinase light chain protein and uses thereof

Abstract

The present invention provides a synthetic gene encoding the human enterokinase light chain protein, a recombinant vector comprising the synthetic gene encoding the protein, a plant cell transformed with the recombinant vector, a method for producing the human enterokinase light chain protein in a plant by using the recombinant vector, a method for producing a plant producing the human enterokinase light chain protein by transforming a plant cell with the recombinant vector, a plant producing the human enterokinase light chain protein which is produced by the method, and a seed thereof, and a composition for large-scale production of the human enterokinase light chain protein in a plant, in which the composition comprises the synthetic gene encoding the human enterokinase light chain protein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY

[0001] This patent application is a National Phase application under 35 U.S.C. §371 of International Application No. PCT / KR2012 / 005757, filed Jul. 19, 2012, which claims priority to Korean Patent Application No. 10-2012-0036227, filed Apr. 6, 2012, entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present invention relates to a plant producing the human enterokinase light chain protein and uses thereof. More specifically, it relates to a synthetic gene encoding the human enterokinase light chain protein, in which codon usage is optimized for expression in a plant, a recombinant vector comprising the synthetic gene encoding the protein, a plant cell transformed with the recombinant vector, a method for producing the human enterokinase light chain protein in a plant by using the recombinant vector, a method for producing a plant producing the human enterokinase light chain protein by trans...

Examples

example 1

Gene Synthesis

[0070]To the primer shEK-F1 (SEQ ID NO: 4) of Table 1, shEK-R1 (SEQ ID NO: 5), shEK-R2 (SEQ ID NO: 6), shEK-R3 (SEQ ID NO: 7), shEK-R4 (SEQ ID NO: 8), and shEK-R5 (SEQ ID NO: 9) were linked in order by a polymerization reaction. Also, to the primer shEK-F2 (SEQ ID NO: 10), shEK-R6 (SEQ ID NO: 11), shEK-R7 (SEQ ID NO: 12), shEK-R8 (SEQ ID NO: 13), shEK-R9 (SEQ ID NO: 14), and shEK-R10 (SEQ ID NO: 15) were linked in order by a polymerization reaction. Then, by a polymerization reaction of each fragment, the DNA fragment of 705 bp, which corresponds to a synthetic gene encoding the human enterokinase light chain protein with optimized rice codon, was obtained (SEQ ID NO: 1). It was then cloned into pGEM-T Easy vector and used for preparing pJKFEK which contains the synthesized gene encoding the human enterokinase light chain protein. As a result of determining the nucleotide sequence, correct synthesis was confirmed. For having extra-cellular secretion of the human entero...

example 2

Construction of Plant Expression Vector

[0071]pJKF3DEK was digested with the restriction enzymes BamHI and KpnI. After isolating by agarose gel electrophoresis the gene fragment encoding the human enterokinase light chain protein, which includes the secretion signal for rice alpha amylase 3D (RAmy3D) of 857 bp, it was introduced to the plant expression vector pMYN75 after digestion with the restriction enzymes BamHI and KpnI. As a result, the plant expression vector pJKF39 for producing the human enterokinase light chain (HEKL) protein was produced. Further, pJKF3DEKHis was digested with the restriction enzymes BamHI and KpnI. After isolating by agarose gel electrophoresis the gene fragment encoding the human enterokinase light chain protein, which includes the secretion signal for rice alpha amylase 3D (RAmy3D) of 877 bp, it was introduced to the plant expression vector pMYN75 obtained by digestion with the restriction enzymes BamHI and KpnI. As a result, the plant expression vector...

example 3

Transformation of Plant

[0072]pJKF39 and pJKF40 were introduced to rice callus by using PDS-1000 / He Biolistic Particle Delivery system of Bio-Rad Laboratories, Inc. and Agrobacterium transformation method. After the inoculating for 10 minutes the Agrobacterium tumefacience comprising the recombinant vector which has been produced to introduce the gene encoding enterokinase light chain protein to the rice callus, in vitro culture was performed for three days. After the in vitro culture, sub-culture was performed on a selection medium added with hygromycin. As a result, it was possible to confirm the normal growth of the callus. In addition, by using 67 lines in total including the plant transformed by gene gun method, the transformed pJKF39 line obtained by using (Agrobacterium) (pJKF39 a1˜9), the transformed pJKF39 line obtained by gene gun method (pJKF39 b1˜30), the transformed pJKF40 line obtained by using Agrobacterium (pJKF40 a1˜12), and the transformed pJKF40 line obtained by ge...

CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY

[0001] This patent application is a National Phase application under 35 U.S.C. §371 of International Application No. PCT / KR2012 / 005757, filed Jul. 19, 2012, which claims priority to Korean Patent Application No. 10-2012-0036227, filed Apr. 6, 2012, entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present invention relates to a plant producing the human enterokinase light chain protein and uses thereof. More specifically, it relates to a synthetic gene encoding the human enterokinase light chain protein, in which codon usage is optimized for expression in a plant, a recombinant vector comprising the synthetic gene encoding the protein, a plant cell transformed with the recombinant vector, a method for producing the human enterokinase light chain protein in a plant by using the recombinant vector, a method for producing a plant producing the human enterokinase light chain protein by trans...

Examples

example 1

Gene Synthesis

[0070]To the primer shEK-F1 (SEQ ID NO: 4) of Table 1, shEK-R1 (SEQ ID NO: 5), shEK-R2 (SEQ ID NO: 6), shEK-R3 (SEQ ID NO: 7), shEK-R4 (SEQ ID NO: 8), and shEK-R5 (SEQ ID NO: 9) were linked in order by a polymerization reaction. Also, to the primer shEK-F2 (SEQ ID NO: 10), shEK-R6 (SEQ ID NO: 11), shEK-R7 (SEQ ID NO: 12), shEK-R8 (SEQ ID NO: 13), shEK-R9 (SEQ ID NO: 14), and shEK-R10 (SEQ ID NO: 15) were linked in order by a polymerization reaction. Then, by a polymerization reaction of each fragment, the DNA fragment of 705 bp, which corresponds to a synthetic gene encoding the human enterokinase light chain protein with optimized rice codon, was obtained (SEQ ID NO: 1). It was then cloned into pGEM-T Easy vector and used for preparing pJKFEK which contains the synthesized gene encoding the human enterokinase light chain protein. As a result of determining the nucleotide sequence, correct synthesis was confirmed. For having extra-cellular secretion of the human entero...

example 2

Construction of Plant Expression Vector

[0071]pJKF3DEK was digested with the restriction enzymes BamHI and KpnI. After isolating by agarose gel electrophoresis the gene fragment encoding the human enterokinase light chain protein, which includes the secretion signal for rice alpha amylase 3D (RAmy3D) of 857 bp, it was introduced to the plant expression vector pMYN75 after digestion with the restriction enzymes BamHI and KpnI. As a result, the plant expression vector pJKF39 for producing the human enterokinase light chain (HEKL) protein was produced. Further, pJKF3DEKHis was digested with the restriction enzymes BamHI and KpnI. After isolating by agarose gel electrophoresis the gene fragment encoding the human enterokinase light chain protein, which includes the secretion signal for rice alpha amylase 3D (RAmy3D) of 877 bp, it was introduced to the plant expression vector pMYN75 obtained by digestion with the restriction enzymes BamHI and KpnI. As a result, the plant expression vector...

example 3

Transformation of Plant

[0072]pJKF39 and pJKF40 were introduced to rice callus by using PDS-1000 / He Biolistic Particle Delivery system of Bio-Rad Laboratories, Inc. and Agrobacterium transformation method. After the inoculating for 10 minutes the Agrobacterium tumefacience comprising the recombinant vector which has been produced to introduce the gene encoding enterokinase light chain protein to the rice callus, in vitro culture was performed for three days. After the in vitro culture, sub-culture was performed on a selection medium added with hygromycin. As a result, it was possible to confirm the normal growth of the callus. In addition, by using 67 lines in total including the plant transformed by gene gun method, the transformed pJKF39 line obtained by using (Agrobacterium) (pJKF39 a1˜9), the transformed pJKF39 line obtained by gene gun method (pJKF39 b1˜30), the transformed pJKF40 line obtained by using Agrobacterium (pJKF40 a1˜12), and the transformed pJKF40 line obtained by ge...

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